2.5.-Analytical methods
2.5.1.- Cell number
Cell number was determined by manual counting using a Neubauer hemocytometer and a phase contrast microscope (Nikon eclypse TS100). Cell viability was determined by the trypan blue dye exclusion method (1:1 mixture of a 0.2% trypan blue (Gibco ThermoFisher Scientific) in phosphate buffer saline and cell sample).
2.5.2.- Metabolites concentration
For the metabolite analysis, samples were centrifuged at 3000g for 3 minutes (Spectrafuge) and the pellet was discarded. Daily measurements of centrifuged samples were obtained for glucose, lactate, glutamine, glutamate, ammonia, K+, Na+ and osmolality using the BioProfile 400 (Nova Biomedical, USA).
Prior the amino acid analysis, supernatants were filtrated using 96-well filter plates (AcroPrep Advance, 0.2 µm Supor Short Tip Natural PP, Pall Corporation). Amino acids were quantified using a method based on Valgepea et al. (2017) with the following modifications: amino acids were derivatized in the HPLC autosampler (Dionex Ultimate 3000) and the sample was injected into an AdvanceBio AAA (2.7 um, 4.6 x 100 mm, Agilent PN: 655950-802) with a guard column (AdvanceBio AAA 2.7 um, 4.6 x 5 mm PN: 820750-931). The HPLC gradient was 5-22% B from 0-7 min, 22-23% B from 7-8 min, 23-30% B from 8-9 min, 30-40% B from 9-14 min, 40-50% B from 14-16 min, 50-55% B from 16-18 min, 55-100% B from 18-18.5 min and kept at 100% to 20.1 min, decreased to 5% B until 24 min where chromatography finished. Buffer A was 40 mM Na2HPO4, 0.02% NaN3(w/v) at pH 7.8. Buffer B was 45% (v/v) acetonitrile, 45% (v/v) methanol and 10% (v/v) water. Flow rate was 1 mL/min from 0-18.5 min and 1.5 mL/min from 18.5-22.1 min thereafter 1 mL/min to 24 min. Derivatized amino acids were monitored using a fluorescence detector. OPA-derivatized amino acids were detected at 340ex and 450em nm and FMOC-derivatized amino acids at 266ex and 305em nm. Quantifications were based on standard curves derived from serial dilutions of an in-house prepared mixed amino acid standard. The upper and lower limits of quantification were 75 and 0.5 μg/mL, respectively. Chromatograms were integrated using Chromeleon ver 7.1.3.
2.5.3.- Product concentration
Product concentration was measured using Valita® TITER Assay at the moment the cells reached the maximum cell density in bioreactor. Valita® TITER Assay is a fluorescence polarization assay for the quantitative detection of IgG antibody in cell culture. Standard curve was performed using a known concentration IgG standard (Human IgG Isotype Control, ThermoFisher) diluted in the same cell culture media of the real experiments. 8-point standard curve performed by triplicated was used. The Valitacell Evaluation tool software was used for analyzing titer assays.